The overall goal of this project is to understand how small GTPases of the Rho family regulate the stability and organization of microtubules (MTs) during cell polarization. The dynamics of MTs gives them the ability to response to external signals during cell polarization, yet little is known about how these signals are transduced to MTs or the proteins that are involved in MT rearrangements. Cells migration into an in vitro wound is a model system for studying the signals regulating MT as the contribution of soluble, matrix and cell-associated factors can be dissected. In the previous grant period, we found that the two rearrangements of MTs in wound edge migrating fibroblasts, formation of a subset of unusually stable MTs and reorientation of the MT organizing center (MTOC), are both triggered by serum lysophosphatidic acid (LPA), but are separately regulated by Rho and Cdc42 GTPases. Both of these rearrangements are thought to involve interactions of MT ends with the cell cortex, a process termed MT capture. We identified mDia, EB1, APC, GSK3beta and novel PKCs as factors working downstream of Rho and two separate pathways working downstream of Cdc42; one involving Par6, dynein and dynactin maintenance of the MTOC at the cell center, the other involving MRCK, actin and myosin II functioning in a novel reward movement of the nucleus. The current aims are to further explore the mechanism of MT stabilization by: exploring how mDia's activity toward MTs and actin is apportioned, by testing whether mDia can directly affect MT stabilization and whether EB1 and APC may affect mDia's activity toward MTs. We will also test whether complexes between mDia, EB1 and APC are regulated by GSK3beta and and screen for additional proteins that may contribute to MT stabilization. The mechanism of Par6, dynein and dynactin regulated MTOC centration will be explored by determining how Par 6 regulates dynein and dynactin, whether additional proteins regulate dynein and dynactin and whether dynein and dynactin maintain the MTOC at the cell center by cortical MT capture. Understanding how Rho GTPases and the pathways they stimulate act to regulate MTs will provide new information about the fundamental ways cells transduce signals to control cytoskeletal systems during cell migration, a process of importance for development, wound healing and metastasis.